YAP/TAZ‐TEAD signalling axis: A new therapeutic target in malignant pleural mesothelioma

Abstract The Hippo signalling pathway, a highly conserved signalling cassette, regulates organ size by controlling cell growth, apoptosis and stem cell self‐renewal. The tumourigenic potential of this pathway is largely attributed to the activity of YAP/TAZ, which activate the TEAD1‐4 transcription factors, leading to the expression of genes involved in cell proliferation and suppression of cell death. Aberrant regulation of the YAP/TAZ‐TEAD signalling axis is commonly observed in malignant pleural mesothelioma (MPM), an insidious neoplasm of the pleural tissue that lines the chest cavity and covers the lungs with poor prognosis. Given the limited effectiveness of current treatments, targeting the YAP/TAZ‐TEAD signalling cascade has emerged as a promising therapeutic strategy in MPM. Several inhibitors of the YAP/TAZ‐TEAD signalling axis are presently undergoing clinical development, with the goal of advancing them to clinical use in the near future.


| YAP/ TA Z B I OLOGY: S TRUC TURE/ FUN C TION REL ATIONS HIPS
The yes-associated protein (YAP) and the transcriptional coactivator with PDZ-binding motif (TAZ) are transcriptional cofactors, integral components of the Hippo signalling pathway.In humans, the YAP1 gene is located on chromosome 11q22 and encodes the 65-kDa YAP protein.YAP comprises several functional domains, including a transcriptional enhanced associate domain (TEAD)-binding (TB) region, two WW domains, an SH3-binding motif, a coiled-coil domain, a transcription activation domain, an N-terminal proline-rich domain and a C-terminal PDZ-binding motif.There are eight different isoforms of the YAP gene, which are classified into YAP1 and YAP2 subgroups and differ in the WW domain.The TAZ gene is located on chromosome 3q23-q24 and encodes the 43-kDa TAZ protein, which is structurally similar to YAP but has only one WW domain and lacks the SH3-binding motif and the proline-rich domain. 1,2P and TAZ are transcriptional coactivators whose localization alternates between the cytoplasm and the nucleus.Their WW domains interact with PPXY motifs of various transcription factors, and their TB region serves as an interaction site with members of the TEAD family of transcription factors (TEAD1-4), triggering the expression of genes associated with malignant transformation in certain cancer types.YAP and TAZ activity is also regulated by members of the 14-3-3 family of phosphoprotein-binding proteins. 3ey share various transcription factors, but they also maintain their own target transcription factors, such as ErbB4 and p73 for YAP and peroxisome proliferator-activated receptor gamma (PPARγ), paired box gene 3 (Pax3), T-box 5 (TBX5) and thyroid transcription factor-1 (TTF-1) for TAZ. 1
Multiple upstream regulators of the Hippo cascade have been described.Among these is the tumour suppressor protein NF2 (also known as Merlin), which forms a complex with kidney and brain protein (KIBRA; also known as WWC1).Together, they activate MST1/2 or recruit LATS1/2 for activation by MST1/2, ultimately phosphorylating the YAP/TAZ complex. 9The striatin-interacting phosphatase and kinase (STRIPAK) complex dephosphorylates MST1/2, leading to YAP/TAZ activation. 10Thousand-and-one amino acid kinases (TAO) activate MST1/2 through direct phosphorylation, thus inhibiting the transcriptional function of YAP/TAZ 11 (Figure 1).The mitogen-activated kinase kinase kinase kinase (MAP4K) family of kinases contributes significantly to the activation of the Hippo cascade, by directly phosphorylating and activating LATS1/2 (Figure 1).
GPCRs linked with G12/13, Gq/11 or Gi/o proteins, such as lysophosphatidic acid (LPA) and thrombin receptors, trigger the activation of YAP/TAZ.Conversely, GPCRs associated with Gs proteins, such as epinephrine and glucagon receptors, impede the activity of YAP/TAZ. 13chanotransduction, the exchange of physical forces between cells and their extracellular matrix (ECM), is a critical factor regulating YAP/TAZ activity.This exchange is primarily mediated by integrins, which are connected on their cytoplasmic side with the F-actin cytoskeleton through focal adhesions, in a manner involving the integrinlinked kinase (ILK), focal adhesion kinase (FAK) and Src proteins.
Regarding cell-cell adhesion molecules, α-catenin is attached to the cytoplasmic domain of E-cadherin.In the presence of 14-3-3, αcatenin binds to phosphorylated YAP, preventing YAP activation and nuclear translocation. 17Cell-cell adhesion molecules play a role in establishing cell polarity and forming adherens junctions.Adherens junctions, in turn, modulate the Hippo pathway by impacting the localization and activity of components such as NF2. 18Discs large homologue 5 (DLG5) is a protein member of the membrane-associated guanylate kinase (MAGUK) complex, which consists of scaffold molecules for protein complexes containing various receptors and signalling elements on the cell membrane.DLG5 directly interacts with MST1/2 to negatively regulate the activity of the Hippo pathway 19 (Figure 1).Epithelial-mesenchymal transition (EMT) is characterized by loss of cell polarity, disruption of cell-cell junctions and cytoskeletal remodelling.EMT is implicated in carcinogenesis by activating YAP and TAZ and promoting tumour survival and progression. 20clear YAP/TAZ accumulation above a critical threshold is linked to various cancer hallmarks of different cancer types, including malignant pleural mesothelioma (MPM).These encompass traits such as cell proliferation, phenotypic plasticity, resistance to drugs, acquisition of metastatic potential and modulation of the tumour microenvironment through the control of stromal cells, inflammation, senescence, immunity and angiogenesis. 21

| TARG E TING THE YAP/ TA Z-TE AD S I G NALLING A XIS IN MPM
Malignant pleural mesothelioma is an aggressive asbestosassociated thoracic tumour originating from pleural mesothelial cells with a poor prognosis (median survival of MPM patients is a mere 8-14 months), due to late-stage diagnosis and a highly infiltrative phenotype.NF2, a major upstream activator of the Hippo pathway, is commonly mutated in MPM, with 30%-40% of cases exhibiting NF2 inactivation. 22Notwithstanding the progress in diagnostic tools and biomolecular research, therapeutic options for the management of MPM are still limited; therefore, novel therapeutic targets are currently being explored.The overexpression of YAP/TAZ is associated with oncotherapy resistance, leading to the ineffectiveness of the available treatment approaches. 23On this basis, the YAP/TAZ-TEAD signalling axis was recently introduced as a novel therapeutic target in MPM.YAP/TAZ have been characterized as challenging targets for drug development.Nevertheless, there have been efforts to experimentally decrease their expression using RNA-interference (RNAi) methods.ION537, an antisense oligonucleotide (ASO) targeting YAP1 mRNA effectively hindered YAP expression in tumour xenografts 24 and is currently being evaluated in a phase I clinical trial (NCT04659096), enrolling patients with solid malignancies.
TEAD activity depends on palmitoylation, a highly conserved process that involves the attachment of palmitate onto cysteine residues via a thioester linkage. 25Using a mesothelioma xenograft model, Tang et al. targeted palmitoylation with small-molecule compounds and demonstrated that NF2-deficient cancer cells exhibit sensitivity to inhibition of TEAD palmitoylation. 26VT3989 is an orally administered, highly potent and selective inhibitor of TEAD palmitoylation, which blocks YAP function and has shown promising preclinical activity.The first-in-human phase I trial (NCT04665206) of VT3989 enrolled 67 patients who had progressed on prior chemotherapy regimens, 29 of whom had MPM.VT3989 was found to be safe and well tolerated without dose-limiting toxicities.The most common adverse events were proteinuria, albuminuria and peripheral edema, primarily when the drug was administered in a continuous schedule.Seven patients (six with refractory malignant mesothelioma pleural or non-pleural) achieved RECIST v1.1 partial responses. 27Another oral small-molecule inhibitor of TEAD palmitoylation, which is presently in phase I clinical trial evaluation (NCT05228015), is IK-930.In preclinical studies, IK-930 exhibited antitumour effects in mouse xenograft models featuring genetic alterations in the Hippo pathway.Currently, it is being assessed as an oral treatment option for patients with advanced solid tumours. 28here have also been efforts to target YAP/TAZ interaction with TEAD, with the pioneer drug being verteporfin, which however displayed YAP-independent cytotoxic effects. 29IAG933, a potent and direct small-molecule inhibitor that disrupts the interaction between YAP and TEAD proteins, is currently in a phase I clinical trial (NCT04857372) in patients with mesothelioma, NF2/LATS1/2mutated tumours and tumours harbouring functional YAP/TAZ fusions (i.e., YAP/TAZ hybrids that hyperactivate a TEAD-based transcriptome).In preclinical xenograft and primary-tumour derived MPM models, IAG933 demonstrated robust antitumour efficacy. 30ble 1 and Figure 1 present the inhibitors of the YAP/TAZ-TEAD axis that are currently being evaluated in clinical trials.
In the preclinical setting, SWTX-143 is a YAP/TAZ-TEAD axis inhibitor which binds to the palmitoylation pocket of all four TEAD isoforms and causes irreversible TEAD inhibition.In subcutaneous xenograft models with human cells and in an orthotopic mesothelioma mouse model, it was demonstrated that SWTX-143 causes strong mesothelioma regression. 31K-975 is another potent, selective and orally active TEAD inhibitor, with a strong inhibitory effect against protein-protein interactions between YAP1/TAZ and TEAD.
It exhibited strong antitumour activity in preclinical mesothelioma models, but it was associated with renal toxicity which might present challenges in clinical application. 32Lastly, JM7 was found to preclinically inhibit YAP transcriptional reporter activity in NF2mutant mesothelioma cells.JM7 is novel small-molecule inhibitor of YAP activity, which hampers TEAD palmitoylation alongside YAP target-gene expression, without affecting YAP/TEAD localization.
Since YAP activity in cancer cells and immune cells interferes with immunotherapy, 33 JM7 could be combined with immunotherapeutic agents in clinical models in the future. 34ho GTPases and ROCK are also appealing targets. 15Nonetheless, accumulating evidence suggests that YAP/TAZ may also have a tumour-suppressive role depending on the context.Thus, careful consideration is warranted when exploring Hippo signalling as a target in future clinical trials. 35terestingly, it has been shown that sirtuin 1 (SIRT1), a NAD +dependent protein deacetylase, deacetylates YAP2 in hepatocellular carcinoma (HCC) cells and SIRT1-mediated YAP deacetylation increases the YAP2-TEAD4 association, leading to YAP2-TEAD4 transcriptional activation and upregulated cell growth in HCC cells. 36In this vein, an increasing amount of preclinical data highlights the effectiveness of

TA B L E 1
Inhibitors of the YAP/TAZ-TEAD axis currently in clinical trials.
histone deacetylase inhibition in MPM cell lines and mouse xenograft models, 37 suggesting that the combinatorial use of histone deacetylase inhibitors (HDCi), for example, depsipeptide, a HDACi exhibiting antitumour effects against several types of solid tumours, 38 with immunotherapy may provide an additional strategy towards formulating more effective therapeutic regimens in patients with MPM.However, as emphasized for other solid tumour types, such as breast carcinomas with malignant-appearing microcalcifications on mammography, 39,40 stage-specific biomarker 'signatures'/gene expression profiling and immune system status should also be taken into consideration in designing tailored combinatorial therapies for MPM patients.

| CON CLUS ION
The YAP/TAZ-TEAD signalling axis is often dysregulated in multiple

Future
steps in the field could be oriented towards targeting other molecules of the Hippo cascade.Potential therapeutic agents targeting YAP/TAZ include bromodomain and extra-terminal motif (BET) inhibitors, which focus on bromodomain-containing protein 4 (BRD4) and its related proteins, as YAP/TAZ are known to engage BRD4 on chromatin.Statins impede YAP nuclear translocation and augment sensitivity to specific cancer medications.Molecules interfering with the activity of Src and its family members (e.g., dasatinib) have also exhibited YAP/TAZ inhibition in vitro and in vivo.
tumour subtypes, including MPM.Allosteric inhibitors that disrupt the YAP-TEAD interaction have shown promising antitumour efficacy mainly in the preclinical setting.Various broadly acting TEAD inhibitors are presently being developed aiming at advancing them to clinical use as monotherapy or in combination with the existing treatment options in the years to come.The outcomes of the ongoing clinical trials will provide insights into the effectiveness of these inhibitors in managing MPM, potentially opening new avenues for improved overall survival of cancer patients.